Name: an integrated workflow of identification and quantification on fdr control-based untargeted metabolome
Uploaded: 2022-09-20T13:00:00
Description: Watch this Scientific Journal Video about An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome at JoVE.com

本研究由国家重点研发计划（2018YFC0910200/2017YFA0505001）和广东省重点研发计划（2019B020226001）支持。

1. 准备代谢组学数据集进行分析
注意：在本演示中，我们使用不含QC样品的代谢组学数据集。需要病例组和对照组的数据。为了进行演示，我们使用GNPS数据库27中的公共数据集。
<ol><li>转到网页 https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp。单击浏览数据集。</li>
	<li>在 “标题 ”列中搜索关键字“msv000084112”。单击数据?...

<ol>
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罗斯福对非靶向代谢物的控制一直是一个巨大的挑战。在这里，我们展示了一个完整的大规模非靶向代谢组学分析（定性和定量）管道，具有罗斯福控制。这有效地减少了在MS分析中非常常见的误报。
为您的研究准备适当的参考光谱库是一个关键点。成功且灵敏的MS/MS识别不仅需要适当的匹配算法，还需要适当的参考光谱库。由于以下原因，公共光谱库的适用性受到限制：（1...

代谢物在生物过程中起着重要作用。代谢物通常是各种过程的调节剂，如能量转移，激素调节，神经递质调节，细胞通讯和蛋白质翻译后修饰等1，2，3，4。非靶向代谢组学提供了许多代谢物的全球视图5，6。随着质谱和色谱技术的进步，代谢组MS/MS谱的通量近年来迅速增加7，8，9，10，11。为了从这些庞大的数据集中鉴定代谢物，开发了各种注释软件11，例如MZmine12，MS-FINDER13，CFM-ID14，MetFrag15和SLAW16。但是，这些标识通常包含许多误报。原因包括：（1）MS/MS光谱包含随机噪声，这可能会误导峰值匹配。（2）异构体和碎片能量的差异导致多个光谱指纹，从而增加参考库的体积。（3） 参考库的质量各不相同。需要一个适当的标准来建立一个良好的参考光谱库。因此，非目标代谢组学的系统性错误发现率（FDR）控制对于功能代谢组研究7，8，9，17至关重要。
经验贝叶斯方法和目标诱饵策略都普遍解决了罗斯福的控制问题。Kerstin Scheubert等人认为，基于碎片树的方法对诱饵数据库生成的目标诱饵策略是FDR控制9的最佳方法。王旭生等人设计了一种基于化学八位字节规则的诱饵生成方法，提高了FDR估计的精度17。演示了用于生成诱饵数据库的光谱库以获得更好的性能18.在这里，我们改进了基于光谱库的方法，并开发了一种名为XY-Meta19 的软件，可以进一步提高FDR估计的精度。它使用现有的参考光谱库为目标诱饵方案下的 FDR 控制生成诱饵库。XY-Meta 支持自己的光谱匹配和余弦相似性算法。它允许传统的搜索和迭代搜索模式。在 FDR 评估的步骤中，它支持目标诱饵串联模式和分离模式。为了获得更好的灵活性，XY-Meta 接受外部诱饵库。
代谢物的峰检测和定量也是非靶向代谢组分析的重要步骤。峰检测是代谢组鉴定的主要方法。一般而言，代谢物峰检测的准确性受多种因素的影响，如质谱的噪声信号、代谢物丰度低、污染物、代谢物降解产物20等。当样品数量过多或液相色谱柱被非靶向代谢组实验替换时，可能会出现显著的批次效应，这是代谢组定量的主要挑战21、22、23。目前，XCMS24、Workflow4蛋白质25、iMet-Q26和metaX19 等软件可以对非靶向代谢组进行峰检测和定量，但我们建议metaX的管道更完整，更易于使用。在这里，我们演示了使用XY-Meta的公开数据集msv000084112的鉴定和FDR控制过程，以及使用metaX对代谢物的峰检测和定量。此工作流只需要两个组，每个组至少需要两个样本。无论质谱仪平台、电离模式、电荷模式和样品类型如何，都需要MS/MS光谱数据，并且可以支持基于样品的归一化和基于峰的归一化。按照这个例子，研究人员可以以易于处理的方式进行代谢组学鉴定和定量。使用此管道需要 R 编程功能。为了帮助没有任何编程知识的研究人员，我们还开发了一个用于代谢组学分析的云分析平台。我们在 补充材料 5 中演示了此云分析平台。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>GNPS</td>
			<td>open source</td>
			<td>n/a</td>
			<td>https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp</td>
		</tr>
		<tr>
			<td>XY-Meta</td>
			<td>open source</td>
			<td>n/a</td>
			<td>https://github.com/DLI-ShenZhen/XY-Meta</td>
		</tr>
		<tr>
			<td>metaX</td>
			<td>open source</td>
			<td>n/a</td>
			<td>https://github.com/wenbostar/metaX</td>
		</tr>
		<tr>
			<td>ProteoWizard</td>
			<td>Free Download</td>
			<td>3.0.22116.18c918b-x86_64</td>
			<td>https://proteowizard.sourceforge.io/download.html</td>
		</tr>
		<tr>
			<td>CHI.Client</td>
			<td>Free Download</td>
			<td>ndp48-x86-x64-allos-enu</td>
			<td>http://www.chi-biotech.com/technology.html?ty=ypt</td>
		</tr>
	</tbody>
</table>

an integrated workflow of identification and quantification on fdr control-based untargeted metabolome

近年来，非靶向代谢组学技术被广泛使用。然而，快速增加的通量和样品数量会产生大量的光谱，这给质谱光谱的质量控制带来了挑战。为了减少误报，必须进行错误发现率（FDR）质量控制。最近，我们开发了一种用于FDR控制非靶向代谢组鉴定的软件，该软件基于名为XY-Meta的目标诱饵策略。在这里，我们演示了一个完整的分析管道，它将 XY-Meta 和 metaX 集成在一起。该协议展示了如何使用XY-meta从现有参考数据库生成诱饵数据库，并使用Target-诱饵策略在开放获取数据集上进行大规模代谢组鉴定来执行FDR控制。差异分析和代谢物注释是在运行metaX进行代谢物峰检测和定量后进行的。为了帮助更多的研究人员，我们还为这些分析开发了一个用户友好的基于云的分析平台，无需生物信息学技能或任何计算机语言。

msv000084112的原始数据通过ms转换进行转换.exe并生成mgf文件（补充材料S6）。
XY 元生成的 GNPS-NIST14-MATCHES_Decoy.mgf 文件位于 /database 文件夹下。这是从原始参考光谱库GNPS-NIST14-Match.mgf生成的诱饵库。这个诱饵库可以重复使用。重用此诱饵库时，用户应在 parameter.default 文件中将decoy_pattern设置为 1，并将诱饵输入设置为诱饵库的绝对路径。鉴定结果在/mgf文件夹（后缀为.m...

Watch this Scientific Journal Video about An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome at JoVE.com

An Integrated Workflow of Identification and Quantification on FDR Control-Based Untargeted Metabolome

我们构建了一个非靶向代谢组学工作流程，将XY-Meta和metaX集成在一起。在该协议中，我们展示了如何使用XY-Meta从开放获取光谱参考中生成诱饵光谱库，然后进行FDR控制，并在鉴定代谢组学光谱后使用metaX定量代谢物。

基于FDR对照的非靶向代谢组的鉴定和定量集成工作流程

Untargeted metabolomics techniques are being widely used in recent years. However, the rapidly increasing throughput and number of samples create an ...

该方案能够基于FDR质量控制对非靶向代谢组进行定性和定量分析，从而有效减少代谢物鉴定的假阳性。该工作流程集成了XY-Meta，该方法使用靶诱饵策略更准确地评估FDR，以便在定性分析模块中鉴定代谢组。该措施可以有效过滤非靶向代谢组鉴定的假阳性结果，从而提高发现生物标志物或关键分子的稳健性。我们希望研究人员能够理解并掌握用于FDR控制的目标诱饵策略，并且应该尝试使用协议的默认参数多次严格运行该管道。首先转到 GNPS 数据库网页，然后单击浏览数据集。在标题列中搜索关键字，然后单击数据集 ID 号。使用FTP下载数据集，并将原始数据保存在相应的文件夹中。要转换原始数据的格式，请首先安装保护顾问软件。在“保护向导”安装路径下，键入 msconvert。exe，后跟将原始数据格式转换为 mzXML 格式的特定参数。同样，使用毫秒转换。exe 将此数据转换为 MGF 格式，并将其存储在 MGF 文件夹中。要准备代谢物的参考光谱库，请转到GNPS网页。搜索关键字 NIST，单击查看详细信息并下载库。将库保存在数据库文件夹中。下载扎伊元程序。在 config 文件夹下找到参数配置文件，并按照文本协议中所述更改其内容。将内收的类型设置为内收文件夹中的列表。使用命令XY-Meta.exe执行代谢物鉴定和错误发现率控制。下载并安装 metaX 软件包。然后编辑示例列表。txt文件指定样品及其相应的质谱数据，如文本协议中所述。使用随文本协议提供的 R 文件运行脚本，以使用 metaX 软件对模拟组和野生类型组进行定量。检查存储定量分析结果的输出文件夹，例如 PCA 图。接下来，修改R脚本中的参数，以使用代谢物鉴定在定性和定量分析中注释峰，以便整合结果并运行R脚本。定量代谢物箱形图显示，健康样本和疾病样本的一般分布相似，均值波动较小。只有3.39%的代谢物具有超过30%的缺失值。两组样品的主成分分析图均表明，metaX显著提高了CV小于0.3的代谢物比例。来自三种统计测试方法的差异检测代谢物的维恩图显示119种常见代谢物。所有注释代谢物的保留时间和电荷分布的质量绘制，错误发现率小于0.01，显示6种显着且差异检测的代谢物。限制工作流测试的时间非常重要。请记住，不要选择太多样品进行分析，并在每组中至少保留两个样品。该技术能够从独立于数据的采集数据中对代谢鉴定进行质量控制，从而使用基于FDR控制的光谱匹配结果构建更强大的参考光谱谱库。

Research

JoVE Journal

Biochemistry

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

科研

生物化学

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

用体外激酶法鉴定细胞周期蛋白依赖性激酶1特异磷酸化部位

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Quantification of Hypopigmentation Activity In Vitro

低聚活性的体外定量

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Quantification of Coenzyme A in Cells and Tissues

细胞和组织中辅酶A的定量

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

用于大规模核相互作用分析的无标签免疫沉淀质谱工作流程

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

An Integrated Raman Spectroscopy and Mass Spectrometry Platform to Study Single-Cell Drug Uptake, Metabolism, and Effects

一个集成拉曼光谱和质谱平台，用于研究单细胞药物接受、代谢和效果

Cells are known to be inherently heterogeneous in their responses to drugs. Therefore, it is essential that single-cell heterogeneity is accounted for in ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

无目标液相色谱-质量光谱学-小麦粒基代谢代谢学分析

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Evaluating the Effect of Pesticides on the Larvae of the Solitary Bees

评估农药对孤独蜜蜂幼虫的影响

Current ecological risk assessments of pesticides on pollinators have primarily considered only laboratory conditions. For the larvae of solitary bees, ...

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

使用透射电子显微镜定量骨骼肌纤维中的亚细胞糖原分布

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...